Despite substantial progress in prevention and treatment, dental caries, commonly known as tooth decay or cavities, remains one of the most common and costly infectious diseases worldwide. According to the CDC, associated health care costs more than 80 billion dollars annually, and the rates of childhood caries in the United States are rising. Novel, comprehensive strategies are needed to effectively combat caries pathogenesis. Cariogenic bacteria form tenacious biofilms on the surface of teeth known as dental plaque. Supported by R01 DE019452, we have over the past five years generated seminal evidence that biofilm regulatory protein BrpA, a member of the LytR-CpsA-Psr family of cell envelope associated proteins, plays a critical role in regulation of cell envelope biogenesis and biofilm formation by Streptococcus mutans. The overall goals of this continuation are to elucidate how BrpA regulates S. mutans stress tolerance response and biofilm formation, traits critical to pathogenicity of this key etiological agent of human dental caries, and to explore the potential of targeting BrpA in strategy against S. mutans and dental caries. In this study, we will use an integrative approach that includes modern molecular and biochemical techniques, such as yeast two-hybrid system, and traditional animal caries model (i) to elucidate the mechanisms that govern the expression of this important streptococcal protein, (ii) to identify protein(s) tha interact with BrpA and uncover how BrpA regulates S. mutans pathophysiology, and (iii) to determine the effects of disrupting BrpA on the ability of S. mutans to colonize the tooth surface and cause carious diseases. Elucidation of cis- and trans-acting factors involved in regulation of BrpA expression and uncovering of proteins interactive with BrpA and their roles in BrpA-mediated functions will constitute a major breakthrough in our understanding of not only just BrpA, but also the LCP family of proteins in regulation of cellular biology in S. mutans and other Gram-positive bacteria. Such findings are expected to facilitate the formulation/development of novel comprehensive strategies against tooth decay and potentially other infections caused by Gram-positive bacteria.